Pyridoxine-derived B6 vitamers and pyridoxal 5'-phosphate-binding proteins in cytosolic and nuclear fractions of HTC cells.
The nuclear fraction of rat hepatoma-derived HTC cells contained approximately 8% of the total cellular pyridoxal 5'-phosphate. HTC cells were able to metabolize [3H]pyridoxine to coenzymatically active pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. As HTC cells did not have any demonstrable pyridoxine-5'-phosphate oxidase activity, the conversion of pyridoxine to pyridoxal 5'-phosphate must have taken place by a nonconventional route. The ratio of pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate in the nonnuclear fraction of HTC cells was approximately 1:1, whereas in the nuclear fraction it was approximately 17:1, indicating that there was selective acquisition of pyridoxal 5'-phosphate by the nucleus. With the aid of a monoclonal antibody specific for the 5'-phosphopyridoxyl group, it was shown that there was one major pyridoxal 5'-phosphate-binding protein in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved nucleoplasmic extract of HTC cells. This finding was confirmed by radioautography of an SDS-PAGE-resolved nucleoplasmic extract obtained from cells grown in a medium containing [3H]pyridoxine. Isoelectric focusing followed by SDS-PAGE also indicated the presence of one major pyridoxal 5'-phosphate-binding protein in the nucleoplasmic extract of HTC cells having a relatively high isoelectric point (approximately 7). Data were obtained indicating that the protein might exist in a higher molecular weight form, probably a dimer. Currently, these findings constitute virtually all of the available information on vitamin B6 and the cell nucleus.
Meisler
N T
NT
Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
Thanassi
J W
JW
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011736:Pyridoxine / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D011506:Proteins; D011732:Pyridoxal Phosphate; D011736:Pyridoxine